Potentially lethal arrhythmias in rare inherited syndromes (idiopathic ventricular fibrillation and Long QT syndrome) have been associated with defects in depolarizing sodium currents. Delineation of the molecular basis and mechanism of the cardiac sodium channel are therefore essential for an accurate understanding of cardiac ventricular depolarization. The determination of the crystal structure of a bacterial inwardly rectifying K channel was a major advance that provided a framework for testing hypotheses concerning the structure of related channels. Nevertheless, crystal structures have the limitation that movement, a major feature of both fast and slow inactivation, is imperceptible. Thus, this proposal will underline vital approaches to structure-function analysis of both fast and slow inactivation of the cardiac sodium channel with an emphasis on understanding the role of these gating mechanisms in inherited arrhythmias. The main focus of this proposal is to develop a library of human cardiac sodium channel constructs containing two fluorescent proteins for later analysis with Fluorescence Resonance Energy Transfer (FRET). FRET and biophysical analysis will be combined to characterize the molecular mechanism and movements of the cardiac sodium channel. By combining patch clamping with FRET, we will be able to measure distance between different regions of the sodium channel in its different gating states under normal a pathological conditions. Therefore this proposal will test the hypotheses that: 1. a library of functional Nav1.5 constructs containing both CFP and YFP can be generated to study motion in cardiac sodium channel using FRET. 2. The C- terminal region of the cardiac sodium channel is involved in fast inactivation and moves during this gating mechanism. 3. Motion in the outer pore mouth underlies slow forms of inactivation of the channel. 4. Mutations in the cardiac sodium channel that cause Long QT syndrome affect regions of the channel involved in inactivation. This project will generate dynamic insights on the structure of the channel that no other approaches, including crystallization have been able to engender thus far. This information on the structure of the channel will be of tremendous help to understand how mutations in this channel are responsible for arrhythmias. [unreadable] [unreadable] [unreadable]